Human pluripotent stem cells (hPSCs), possess the remarkable ability to differentiate into virtually all somatic cell types in the body (pluripotency), while maintaining proliferative capacity in an undifferentiated state (self-renewal). These unique features of hPSCs provides opportunity to generate transplantable cells and tissue material for treatment of diseases and conditions ranging from diabetes, osteoarthritis, and Parkinson's disease, among many others. As many human injuries and diseases result from cellular defects, including those of a single cell type, replacement with appropriate stem cells, progenitor cells, or in vitro differentiated cells, could lead to novel therapeutic approaches in the clinic. Moreover, whereas live donors, cadaveric or fetal sources have served as sources of transplant material, the self-renewal capacity of pluripotent stem cells further provides a near limitless resource of transplantable material. An additional benefit is that hPSCs can be generated in a patient-specific manner, thereby leading to therapauetic approaches that reduce risk of immunological rejection and tolerance. Thus, there is considerable enthusiasm for regenerative cell transplantation, including immunocompatible hPSCS and patient-specific hPSCs.
One strategy for generation of immunocompatible hPSCs is to facilitate parthenogenesis (i.e., asexual reproduction) from an oocyte to generate blastocysts for hPSC isolation. Briefly, this approach relies on artificially inducing oocytes to undergo meiotic and mitotic processes in the absence of sperm fertilization, leading to a diploid (2 maternal genome) parthenote, which can be cultured further into blastocyst from which hPSCs can be isolated. Using the parthenogenesis approach, the isolated hPSCs are known as parthenote-derived human pluripotent stem cells (pn-hPSCs).
An alternative approach allows for generation of patient-specific hPSCs through somatic cell nuclear transfer (SCNT). Somatic cell nuclear transfer involves isolation of the nucleus of a somatic cell of a donor patient and insertion into an enucleated recipient oocyte. Transfer of the donor nucleus to the recipient oocyte's cytoplasm results in reprogramming of the transferred donor nucleus through silencing of somatic cell genes and activation of embryonic genes. From reconstructed oocytes, one can establish blastocysts in culture to isolate hPSCs from the inner cell mass (ICM). As the result of nuclear transfer the hPSCs (NT-hPSCs) carry the nuclear genetic material of the patient, which are patient-specific. There is significant overlap in technical procedures related both techniques. For example, early attempts to achieve NT-hPSC cell lines resulted in the accidental and first known instance of pn-hPSC cell line generation.
Despite promising advances in parthenogenesis and SCNT techniques, there are significant limitations that, at present, preclude practical clinical application. For example, existing parthenogenesis studies have reported successful oocyte activation in approximately 50% of donor oocytes, but a key limitation appears to be induction of blastocyst formation, which can dip to as low as 15% or less of activated oocytes. As related to nuclear transfer, using conventional techniques, only up to as few as 2% of reconstructed oocytes cross the 8-cell threshold towards blastocyst formation. Together, these limitations can mean less than 10% of donor oocytes are successfully cultivated into pn-hPSCs. Subsequent pn-hPSCs may suffer from other undesirable attributes such as aneuploidy or karyotype instability.
As exact mechanisms allowing for consistent and reproducible oocyte activation and blastocyst formation are presently unclear, obstacles presently limiting generation of pSC lines would certainly be helped by a better understanding of these processes.
For example, recombination events occurring during meiotic stages in parthenogenesis can give rise to variable amounts of zygosity. Therefore, real-time visualization of these events would not only improve success rates for blastocyst formation and pn-hPSC cell line generation, but aid manipulation strategies aimed at achieving specific genetic and immunological characteristics of resulting pn-hPSC cells. Similarly, current SCNT approaches for deriving NT-hPSCs use oocytes from in vitro fertilization clinics. High failure rates, also due to aneuploidy, karyotype instability and/or inefficient nuclear reprogramming, requires greater numbers of oocytes for derivation of NT-hPSCs, and are still limited to generating cells that are not patient-specific. Thus, there is a great need in the art for techniques allowing consistent, efficient generation of immunocompatible and patient-specific hPSC using SCNT and/or parthenogenesis techniques.
Described herein are techniques for establishing immunocompatible and/or patient-specific hPSCs. In one aspect, efficient SCNT and/or parthenogenesis is described using improved techniques. This includes improvements in micromanipulation to reduce cellular damage, poloscope microscopy for real-time visualization without use of harsh staining agents and/or UV light exposure. More importantly, key barriers related to efficient blastocyst generation have been eliminated by application of methylation-altering agents promoting genomic activation within a reconstructed oocyte, along with addition of mitotic structures (e.g., centrioles) from sperm derivatives to promote blastocyst expansion. Application of these improved techniques allows realization of banks of pn-hPSCs expressing common human leukocyte antigen (HLA) haplotypes to provide cells immunologically compatible with wide segments of the population. In all aspects, pn-hPSCs and NT-hPSCs can be grown on animal protein-free culture media, as is vital for clinical applications eventually involving human patients, and provides transplantable cells that are genetically and epigenetically stable, and pluripotent.